A major hurdle for the development of effective cancer vaccines is the inability of most currently available cancer vaccines to induce a robust immune responses against a broad spectrum of tumor antigens. It has been well-established that the induction of T-cell mediated antitumor immune responses is critically dependent on the cross-presentation of tumor antigens by mature dendritic cells. Under normal physiologic conditions, cross-presentation by immature dendritic cells is limited to long-lived proteins that are expressed at a high level. The steady and level cross-presentation of these proteins by host immature dendritic cells often leads to tolerance rather than immunity. Although tumor cells express a large spectrum of short-lived proteins and directly present peptides derived from these proteins, short-lived proteins are not crosspresented by dendritic cells due to the ubiquitin conjugation and rapid degradation of these short-lived proteins by the proteasome of tumor cells. Recently we discovered that short-lived proteins could accumulate as ubiquitin-conjugated proteins and be released from tumor cells as particles (DRibbles). We also discovered that DRibbles could serve as an efficient substrate for the cross-priming of tumor-specific naive T cells and active immunization using dendritic cells loaded with DRibbles could mediate tumor regression in mice bearing established tumors. Our long-term goal is the development of effective cancer vaccines using our novel DRibble vaccine technology. This technology enables cross-priming of tumorspecific T cells that recognize a large spectrum of tumor/tumor-associated antigens including short-lived proteins. This SBIR proposal is aimed to determine the feasibility of DRibble vaccine technology using an in vitro human culture system. In the first aim, we will determine the optimal conditions for DRibble production and characterize DRibbles produced from multiple human cancer cell lines. The second aim will investigate whether DRibbles with encapsulated TLR ligands will promoter a robust production of pro-inflammatory cytokines. The third aim will compare the cross presentation efficiency of DRibbles with or without encapsulated TLR ligands. The fourth aim will determine whether tumor-specific T cells could be primed by DRibbles in vitro from PBL of cancer patients. Accomplishment of these aims will lay the foundation for new clinical trials that employ our novel vaccine technology. [unreadable] [unreadable] [unreadable]